A very precise control of cell proliferation is essential for normal animal development and the deregulation of cell division is a hallmark of cancer. The E2F transcription factors are integral components of the machinery regulating the mammalian division cycle. The role of E2F has been conserved throughout evolution. E2F co- ordinates the transcription of hundreds of genes that are needed for cells to divide and proliferate. In the two decades since E2F was first identified it has been assumed that activation of E2F-dependent transcription inevitably results in the increased synthesis of the encoded proteins. I have recently uncovered evidence of an additional layer of post-transcriptional regulation that acts broadly on a significant fraction of E2F-regulated transcripts, and that suppresses the translation of these mRNAs. Drosophila genetic studies show that two RNA-binding proteins, Pumilio and Nanos, functionally suppress E2F activity in vivo. Molecular studies have confirmed that the regulation of E2F/RB and Pumilio/Nanos proteins are intimately interconnected in both flies and humans, with mRNAs encoding activator E2Fs being suppressed via Pumilio response elements (PREs), and the transcription of Nanos and Pumilio genes being suppressed by E2F and RB family members. An explanation for these connections has emerged from omic studies. Our recent comparison of the transcriptional and proteomic changes associated with RB- mutation in mouse lung tissue revealed that although the transcription of E2F target genes were dramatically increased upon RB mutation, very few of these genes displayed protein level changes. Strikingly, an unbiased analysis of the post-transcriptional motifs within mRNAs that are transcriptionally upregulated in RB mutant tissues, without a similar increase in protein levels, found that PRE-motifs are the most significantly enriched. Furthermore analysis of mRNA sequences upregulated in retinoblastoma tumors shows that mRNAs from >50% of E2F-regulated genes contain putative PRE motifs. This proposal will test the hypothesis that Pumilio and Nanos proteins provide a tier of post- transcriptional regulation that acts on a broad swath of E2F-induced transcripts. I will determine which E2F- induced transcripts are suppressed in this manner, when they are regulated, and will investigate the impact of this regulation on protein levels. Deregulated E2F activity drives the proliferation of many human cancers. I will discover whether the control mediated by Pumilio and Nanos buffers RB mutant cells from increases in E2F-dependent transcription, and I will determine whether this apparatus is preserved or lost in cancer cells. Collectively these experiments will define a novel regulatory mechanism that limits the expression of E2F- transcibed genes.